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首页> 外文期刊>Physical chemistry chemical physics: PCCP >Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors
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Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors

机译：基于线辅助光学系统的微秒分辨单分子FRET时间序列测量结合混合光电探测器

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摘要

Single-molecule (sm) fluorescence time series measurements based on the line confocal optical system are a powerful strategy for the investigation of the structure, dynamics, and heterogeneity of biological macromolecules. This method enables the detection of more than several thousands of fluorescence photons per millisecond from single fluorophores, implying that the potential time resolution for measurements of the fluorescence resonance energy transfer (FRET) efficiency is 10 mu s. However, the necessity of using imaging photodetectors in the method limits the time resolution in the FRET efficiency measurements to approximately 100 mu s. In this investigation, a new photodetector called a hybrid photodetector (HPD) was incorporated into the line confocal system to improve the time resolution without sacrificing the length of the time series detection. Among several settings examined, the system based on a slit width of 10 mu m and a high-speed counting device made the best of the features of the line confocal optical system and the HPD. This method achieved a time resolution of 10 ms and an observation time of approximately 5 ms in the sm-FRET time series measurements. The developed device was used for the native state of the B domain of protein A.
机译：基于线路共聚焦光学系统的单分子（SM）荧光时间序列测量是对生物大分子的结构，动力学和异质性进行调查的强大策略。该方法能够从单个荧光团检测超过数千个荧光光子的荧光光子，这意味着用于测量荧光共振能量转移（FRET）效率的潜在时间分辨率是10μs。然而，在该方法中使用成像光电探测器的必要性将空隙效率测量的时间分辨率限制为大约100μm。在该研究中，将称为混合光电探测器（HPD）的新光学探测器纳入线路共焦系统中，以改善时间分辨率而不牺牲时间序列检测的长度。在检查的几个设置中，基于10μm和高速计数装置的狭缝宽度的系统成为了线路共聚焦光学系统和HPD的特征。该方法实现了10ms的时间分辨率和在SM - FRET时间序列测量中的观察时间约为5ms。开发装置用于蛋白质A的B域的天然状态。

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